Abstract
INTRODUCTION
UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamime kinase (GNE) is a bifunctional rate-limiting enzyme in the biosynthesis of sialic acid. Mutations in GNE gene have been linked to sialuria, a rare dominant metabolic disease and to quadriceps sparing myopathy/hereditary inclusion body myopathy type 2, an autosomal recessive disorder which is the same disease as the earlier described distal myopathy with rimmed vacuoles (DMRV) or Nonaka myopathy (OMIM#605820) [1–6].All these definitions have now been replaced after an international consortium by the new name “GNE-myopathy” [7]. So far, 147 different GNE mutations located in the epimerase or the kinase domain or both, have been identified worldwide providing evidence that GNE-myopathy occurs in other than Middle Eastern or Japanese ethnicities in which founder effects are found to account for the higher prevalence [3, 8–10].
The disease is mainly characterized by an early adult-onset progressive distal myopathy with early selective involvement of anterior tibial muscles and sparing of quadriceps even in advanced stages [11]. Thigh and hand muscles are frequently involved in the course of the disease and the median time to loss of ambulation is 17 years after the onset of first symptoms [11, 12]. The most characteristic feature on muscle biopsy is the presence of rimmed vacuoles in muscle fibers showing acid phosphatase activity, while grouped fiber atrophy and tubulofilamentous inclusions without evidence of inflammation are also revealed in most cases of affected individuals [11].
We report the clinical and molecular findings of two affected members of the same family identified with GNE-myopathy, who present with atypical phenotypic features, and compare them with patients carrying the same mutation reported in the literature.
CASE REPORTS
Patient 1
A 32 year-old romani man presented at the age of 26 with a progressively increasing walking difficulty of six years duration. The patient used to be a skillful football player until late adolescence. His first symptoms appeared by the age of 20 and consisted of easy fatigability and difficulty in running and walking long distances.
On presentation at the age of 26, the patient had a steppage gait due to severe bilateral foot drop (foot dorsiflexion 2/5 as defined by MRC scale), while he had also severe bilateral plantar flexion weakness (2/5) and he was totally unable to walk on toes. He had also a milder difficulty in rising from a chair or squatting position. Bilateral muscle wasting and weakness were evident in the lower legs and in first dorsal interosseous muscles. A mild symmetrical proximal muscle weakness of lower limbs was also found with sparing of hip flexion and knee extension. Creatine kinase (CK) was mildly elevated in consecutive measurements and electromyography (EMG) demonstrated myopathic changes with spontaneous activity mainly in distal muscles Muscle biopsy of left biceps brachii revealed fiber size variation and rimmed vacuoles, while a large number of fibers contained prominent dense granular basophilic inclusions (Fig. 1A), not reactive for myotilin or DNAJB6 antibodies. Interestingly, the specimen also showed one single site of perivascular inflammation consisting predominantly of CD8+ lymphocytes and few histiocytes. Computed Tomography (CT) of limb muscles at the age of 29yrs showed prominent fatty degeneration in all lower leg muscles. Hamstring muscles were also affected, while quadriceps was much less involved (Fig. 2A).
The patient is now at age 32 wheelchair bound and he has also severe weakness and atrophy of the intrinsic hand muscles.
Patient 2
A now 32 year old romani male, who is a second cousin of patient 1, was referred to our hospital two years ago for progressive gait instability since the age of 28. Despite the walking difficulties, the patient still remains ambulant. At the first examination, there was a severe and mildly asymmetrical bilateral weakness of plantar flexors (2+/5 at right and 2/5 at left). Ankle dorsiflexion was mildly affected (4+/5 bilaterally). Quadriceps strength was totally intact, while there was a moderate knee flexion weakness (4/5 bilaterally). There was no weakness of upper limbs. CK was always mildly elevated and EMG revealed myopathic changes with some spontaneous activity in tibialis anterior and gastrocnemius medialis muscles bilaterally. Left tibialis anterior muscle biopsy showed extensive pathology with muscle fiber variation rimmed and non-rimmed vacuoles and diffuse basophilia in many highly abnormal degenerative fibers (Fig. 1B). Atrophic vacuolated fibers showed dense aggregate immunostaining with p62, TDP43, ubiquitin and autophagosomal LC3 (Fig. 3). Muscle CT scanning of lower limbs at the age of 30yrs showed prominent fatty degenerative change in the posterior compartments of both thigh and lower legs with sparing of anterior compartment muscles (Fig. 2B).
Both patients were submitted to pulmonary and cardiological assessments which were normal.
Genetic analysis
The pedigree suggested a recessive mode of inheritance. Haplotype linkage of the GNE locus on the chromosome 9 showed shared alleles on both chromosomes in both patients was compatible with GNE being a good candidate. Subsequent Sanger sequencing of the gene showed that both patients were homozygous for the previously reported pathogenic mutation c.1853T>C in exon 11, leading to the p.I618T substitution of isoleucine by threonine in the kinase domain of GNE, according to the new nomenclature (number of reference transcript: NM_001128227) [7].
DISCUSSION
The current report presents two male GNE-myopathy patients of romani ethnic origin from the same family with an early adult onset distal myopathy characterized by coexisting atypical calf involvement. Patient 1 presented with severe difficulty in walking on toes and heels since 20 years of age, and muscle weakness progressed more proximally to involve knee flexors. At the age of 32, he has already been wheelchair bound for the last two years. Patient 2 also developed ankle weakness but with plantar flexion weakness and not dorsiflexion weakness as the lead finding.
The list of differential diagnosis for distal myopa-thies is increasingly expanding and is expected to grow further with the advance in molecular genetics and the subsequent identification of new responsible genetic defects. Moreover, a predominant distal pattern of weakness and atrophy may be encountered in various different muscle diseases which must be included in the diagnostic work-up [13]. Based on the apparent recessive inheritance, early adult onset, quadriceps sparing on muscle imaging and rimmed vacuolar pathology, GNE-myopathy was a definite candidate according to the published diagnostic algorithms [13]. Haplotype linkage and gene sequencing eventually led to the diagnosis of GNE myopathy. The two probands were proven homozygous for the already known pathogenic mutation I618T in the kinase domain of GNE, attributed to remote consanguinity in the family because of the identical haplotypes and a common recent ancestry. This missense mutation has been initially identified in two patients from Italy and USA, respectively, who developed their first symptoms in mid-twenties. The first symptom in those patients was foot drop, and the Italian patient had also proximal weakness with just partial quadriceps sparing [4, 14]. Recently, this specific mutation was found in homozygosity in 50 Bulgarian Roma patients and was also reported as the second most common mutation among GNE patients in Western India. The initial symptom in almost all these patients was foot drop in their third decade [10]. Even more atypically, both our patients presented with bilateral severe weakness also of foot plantar flexion and on muscle imaging the severe dystrophic changes on calf muscles were clearly confirmed without change in the anterior compartment in patient 2. The course of the disease was quite different between these second cousins, with an earlier onset of symptoms, more rapid progression and early disability in patient 1 compared to patient 2. This observation adds further evidence of a possible clinical variability even among related patients, as has been previously reported in Roma patients carrying the same genotype [10].
To date, no clear genotype-phenotype correlation has been reported in patients with GNE-myopathy, despite some diversity of the phenotypic spectrum, including limb girdle involvement, facial weakness and/or hand muscle atrophy [15–18]. The atypical early calf muscle involvement in both patients and the sparing of anterior lower leg muscles in patient 2 emphasizes the clinical heterogeneity and needs to be considered in the differential diagnostic work-up because of disparity with current algorithm schemes. The reason for the variability is not known, but other factors, such as modifier genes or environmental parameters might contribute. Nevertheless, homozygous mutations in the kinase domain are considered to cause a more severe, although rather typical, phenotype compared to both homozygous epimerase domain mutations or compound heterozygous epimerase and kinase domain mutations, which may be associated with a relatively milder course and some infrequent clinical manifestations [12, 15].
Muscle biopsy in both patients showed myopathic changes and prominent vacuoles rimmed by basophilic granular material that stained positive for the autophagy markers LC3, p62 and ubiquitine and the protein aggregation marker TDP-43, which is also a major component of inclusions in ALS and/or FTD and in patients with sporadic inclusion body myositis [19, 20], indicating a possible common degenerative pathways in GNE-myopathy, as well. Although the absence of inflammation is considered the key histological characteristic to distinguish GNE-myopathy from sporadic inclusion body myositis, there are isolated GNE-myopathy cases with some inflammatory infiltrates [3, 21], such as in our patient 1. A clear rimmed vacuolar pathology identified in a patient under age 30 the age may be a more reliabledistinguishing feature.
Definitive curative treatment for GNE-myopathy is still lacking. However, some therapeutic approaches involving sialic acid supplementation or its precursor ManNac showed promising results in mouse models [4, 22], and human trials are underway. Given the variable clinical expression of the disease and thepossible imminent new therapy in the forthcoming years, a high level of diagnostic awareness is required even in patients with atypical clinical features or without a positive family history.
CONFLICT OF INTEREST
The authors have no conflict of interest to report.
Footnotes
ACKNOWLEDGMENTS
The study was supported by research grants from the Juselius Foundation and the Finnish Academy.